Proteases hydrolyze specific peptide bonds in proteins. The residues at the active site are used to classify proteases (Rawlings & Barrett, 1995). Proteases that hydrolyze peptide bonds using metal ions are referred to as metalloproteases (“MP”). The metalloproteinases may be one of the older classes of proteases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures, but many contain a zinc ion. In some cases, zinc may be replaced by another metal such as cobalt or nickel.
The gene and protein of the present invention codes for a human protease belonging to the peptidase M10 family (see Rawlings & Barrett, 1995 for review of protease familial classification). This family contains the sequence . . . HE[ILF]GHXXGLXH . . . (SEQ ID NO:81), which is thought to contain amino acids (histidines and or glutamic acid) which coordinate metal ion binding. Such metal ion coordination facilitates catalysis through the stabilization of a noncovalent, tetrahedral intermediate after the attack of a metal-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group. Metal ion coordination is thought to stabilize the negative charges formed within the active site of the enzyme during catalysis. Such stabilization lowers the transition state energy requirements, and thus results in significant rate enhancements during enzymatic catalysis over non-metal ion coordination conditions (Fersht, A., “Enzyme Structure and Mechanism”, 2nd edition, W.H. Freeman and Company, New York, 1985).
Another important feature of peptidase M10 family is a highly conserved octapeptide ( . . . PRC[GN]XP[DR][LIVSAPKQ] . . . (SEQ ID NO:82)) that has been shown to be involved in autoinhibition of metalloproteases (Breathnach R. et al, 1988; Navre M. et al. 1991). A cysteine within the octapeptide chelates the active site zinc ion, thus inhibiting the enzyme. Between the autoinhibitive octapeptide and the catalytic peptide resides a pair of conserved basic amino acids [RK][RK] that serves as the putative cleavage site for activation by furin proteinases.
The prototype of this family is a human secreted interstitial collagenase called matrix metalloproteinase 1. Substrate proteins for the matrix metalloproteinase 1 include the interstitial collagen group—types I, II, III and alpha-macroglobulins (Vincenti MP et al, 1996). A metalloproteinase gene (XMMP) transiently expressed in Xenopus laevis early embryo development has been discovered (Yang M, Murray M T, Kurkinen M, 1997). It is undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo, suggesting that XMMP plays a role in Xenopus early development. The human MMP-29 gene described herein represents the human ortholog of the Xenopus XMMP.
Metalloproteinases in Disease
Limited-proteolysis by metalloproteases plays a central regulatory role in many physiological and pathophysiological processes. There are many examples of inhibitors of metalloproteases that are useful medications in the treatment of hypertension, heart failure, various forms of cancer and other diseases.
Metalloproteases play many important biological roles in the nervous system, including the spinal cord. There is a balance between the synthesis and degradation of extracellular matrix proteins in the process of synapse formation during development and regeneration. The timing of MP activation is therefore potentially critical. Some MPs have been shown to be upregulated in the spinal cord either during development or in pathological states such as multiple sclerosis, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis. Since MPs degrade extracellular matrix proteins, they would be toxic to developing neurons that depend upon the matrix proteins for survival, neurite outgrowth, and synapse formation. Degradation of the matrix proteins would also cause the breakdown of the blood brain barrier and infiltration of immune cells into the CNS, which occurs in inflammatory conditions such as MS. Other biological processes that metalloproteinases are involved in include fibrillogenesis, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions and ulceration.
Using the above examples, it is clear the availability of a novel cloned metalloproteinase provides opportunities for adjunct or replacement therapy, and are useful for the identification of metalloproteinase agonists, or stimulators (which might stimulate and/or bias metalloproteinase action), as well as, in the identification of metalloproteinase inhibitors. All of which might be therapeutically useful under different circumstances. The metalloproteinase of the present invention can also be used as a scaffold to tailor-make specific metalloproteinase inhibitors.
The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length MMP-29 gene and its encoded polypeptide. Also provided are polypeptide alignments illustrating the strong conservation of the MMP-29 polypeptide to other known metalloproteinases. Data is also provided illustrating the unique tissue expression profile of the MMP-29 polypeptide in testis tissues, which has not been appreciated heretofore.
The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the MMP-29 polypeptide. Such compounds are potential inhibitors of MMP-29 or its homologues. The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of MMP-29 or its homologues.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of MMP-29 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MMP-29 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.